A serum-free, partly defined medium has been devised for the axenic cultivation of Entamoeba including the human pathogen E. histolytica, and the reptilian pathogen E. invadens. A critical component is a heat stable, low-molecular weight, growth factor(s) isolated from casein. Highly repeated DNA of E. histolytica was cloned in a plasmid vector and shown to code for ribosomal RNA by its strong homology with Plasmodium berghei ribosomal DNA and by Northern blot analysis. Subfragments of the highly repeated DNA generated by restriction enzyme treatment were highly specific for E. histolytica hybridizing with DNA from several strains of this species, but not with any of seven other species of Entamoeba tested.